Proteases play essential roles in many disease processes such as Alzheimer's, hypertension, inflammation, apoptosis, and AIDS. Compounds that block or enhance their activity have potential as therapeutic agents. Because the normal substrates of peptidases are linear peptides and because established procedures exist for making non-peptidic analogs, compounds that effect the activity of proteases are natural subjects of combinatorial chemistry. Screening compounds produced by combinatorial chemistry requires convenient enzymatic assays.
The most convenient existing assays for proteases are based on fluorescence resonance energy transfer from a donor fluorophore to a quencher placed at opposite ends of a short peptide chain containing the potential cleavage site. Knight C G, "Fluorimetric assays of proteolytic enzymes," Methods in Enzymol. (1995) 248:18-34. Proteolysis separates the fluorophore and quencher, resulting in increased intensity in the emission of the donor fluorophore. Existing protease assays use short peptide substrates incorporating unnatural chromophoric amino acids, assembled by solid phase peptide synthesis. However, solid phase synthesis poses certain problems of effort and expense.
It is useful to perform enzymatic assays in vivo, in order to more closely mimic conditions in which intracellular proteases act. Conventional artificial substrates prepared by solid-phase synthesis would require microinjection into individual cells, which is impractical as a high-throughput screen. Also, short unfolded peptides are generally rapidly degraded by nonspecific mechanisms inside cells.
The Edans fluorophore is the current mainstay of existing fluorometric assays. Fluorophores with greater extinction coefficients and quantum yields are desirable. The Edans fluorophore often is coupled with a non-fluorescent quencher such as Dabcyl. However, assays performed with such agents rely on the absolute measurement of fluorescence from the donor. This amount is contaminated by other factors including turbidity or background absorbances of the sample, fluctuations in the excitation intensity, and variations in the absolute amount of substrate.